Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full length chimeric TBE/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C), premembrane (M), envelope (E), or nonstructural protein NS1 for the corresponding sequences of DEN4. Full length RNA transcripts prepared from these cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared to DEN4, chimeric TBE(ME)/DEN4 virus grew to higher titer and produced larger plaques in simian cells. In contrast, chimeric TBE(ME)/DEN4 virus produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in TBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, TBE(ME)/DEN4 entered simian cells efficiently and its viral RNA was replicated more rapidly in these cells than was parental DEN4 viral RNA. Following intracerebral inoculation TBE(ME)/DEN4 virus caused fatal encephalitis in both suckling and adult mice, while mice inoculated by the same route with DEN4 did not develop disease. However, unlike wild-type TBEV, chimeric TBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 103 LD50 of TBEV, while mice previously inoculated with DEN4 were not protected. These findings indicate that: (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistant to lethal TBEV challenge, and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a TBE/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a new strategy for the development of live attenuated TBEV vaccines.